Inhibition of TAK1 and/or JAK can rescue impaired chondrogenic differentiation of human mesenchymal stem cells in osteoarthritis-like conditions.

OBJECTIVE: To rescue chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in osteoarthritic conditions by inhibition of protein kinases. METHODS: hMSCs were cultured in pellets. During early chondrogenic differentiation, these were exposed to osteoarthritic synovium-conditioned medium (OAS-CM), combined with the Janus kinase (JAK)-inhibitor tofacitinib and/or the transforming growth factor ß-activated kinase 1 (TAK1)-inhibitor oxozeaenol. To evaluate effects on chondrogenesis, the glycosaminoglycan (GAG) content of the pellets was measured at the time that chondrogenesis was manifest in control cultures. Moreover, mRNA levels of matrix molecules and enzymes were measured during this process, using real-time polymerase chain reaction (RT-PCR). Initial experiments were performed with hMSCs from a fetal donor, and results of these studies were confirmed with hMSCs from adult donors. RESULTS: Exposure to OAS-CM resulted in pellets with a much lower GAG content, reflecting inhibited chondrogenic differentiation. This was accompanied by decreased mRNA levels of aggrecan, type II collagen, and Sox9, and increased levels of matrix metalloproteinase (MMP)1, MMP3, MMP13, ADAMTS4, and ADAMTS5. Both tofacitinib (JAK-inhibitor) and oxozeaenol (TAK1 inhibitor) significantly increased the GAG content of the pellets in osteoarthritis (OA)-like conditions. The combination of both protein kinase inhibitors showed an additive effect on GAG content. In agreement with this, in the presence of OAS-CM, both tofacitinib and oxozeaenol increased mRNA expression of sox9. The expression of aggrecan and type II collagen was also up-regulated, but this only reached significance for aggrecan after TAK1 inhibition. Both inhibitors decreased the mRNA levels of MMP1, 3, and 13 in the presence of OAS-CM. Moreover, oxozeaenol also significantly down-regulated the mRNA levels of aggrecanases ADAMTS4 and ADAMTS5. When combined, the inhibitors caused additive reduction of OA-induced MMP1 mRNA expression. Counteraction of OAS-CM-induced inhibition of chondrogenesis by these protein kinase inhibitors was confirmed with hMSCs of two different adult donors. Both tofacitinib and oxozeaenol significantly improved GAG content in cell pellets from these adult donors. CONCLUSIONS: Tofacitinib and oxozeaenol partially prevent the inhibition of chondrogenesis by factors secreted by OA synovium. Their effects are additive. This indicates that these protein kinase inhibitors can potentially be used to improve cartilage formation under the conditions occurring in osteoathritic, or otherwise inflamed, joints.